E-box promoter motif is required for melatonin to shift SCN neuronal activity rhythms at CT 10.

<p><b>A)</b> The spontaneous electrical activity rhythm in SCN brain slices peaks at CT 6.38 ± 0.13 in controls (<i>n = 3</i>). The dotted line indicates the mean time-of-peak for untreated slices. Large, vertical boxes represent subjective night, CT 12–14. <b>B)</b> At CT 10, MEL (1 nM, 10 min) advances the electrical activity rhythm by 3.6 h ± 0.10 (<i>n = 3</i>). Arrow = time of melatonin treatment. <b>C)</b> E-box decoy ODN has no significant effect on the time-of-peak electrical activity (<i>n = 3</i>). Small box = duration of ODN exposure. <b>D)</b> The MEL-induced phase advance is blocked by the E-box decoy ODN (<i>n = 3</i>). <b>E)</b> Missense ODN has no effect on the MEL-induced advance in time-of-peak electrical activity (<i>n = 3</i>). <b>F)</b> Missense ODN does not block the MEL-induced phase advance at CT 10 (<i>n = 3</i>). <b>G)</b> Summary of the effects of ODN on MEL-induced phase advances at CT 10. **indicates statistically significant difference compared to controls (p ≤ 0.001) as determined by 1-way ANOVA with Tukey’s <i>post- hoc</i> analysis.</p

The PKC inhibitor, chelerythrine chloride, blocks the increase of <i>Per1</i> and <i>Per2</i> mRNA induced by melatonin applied at CT 10.

<p>Pre-treatment with 0.25 mM of the PKC inhibitor, chelerythrine chloride, blocks the melatonin-induced increase in <i>Per1</i> <b>(A)</b> and <i>Per2</i> <b>(B)</b> transcripts after 120 min. Data are shown as percent change of relative mRNA levels compared to control ± SEM, <i>n</i> = 3/condition (** p ≤ 0.01, *p ≤ 0.05, 1-way ANOVA, Tukey’s <i>post-hoc</i> analysis). Controls were exposed to sham treatment lacking MEL. MEL = melatonin. CC = chelerythrine chloride.</p

At CT 6, melatonin does not change the levels of <i>Per1</i> and <i>Per2</i> transcripts, although <i>Bmal1</i> is reduced at 120 min.

<p>Melatonin applied at CT 6 has no significant effect on the expression levels of <i>Per 1</i>, <i>Per2</i>, or <i>Bmal1</i> mRNA after 30 min <b>(A)</b>. After 120 min <b>(B)</b>, only <i>Bmal1</i> mRNA significantly decreases following initiation of melatonin treatment at CT 6. Data are shown as percent change of relative mRNA levels compared to control ± SEM, <i>n</i> = 3–9 /condition, p ≥ 0.05 (<i>Per 1</i>, <i>Per 2</i>), *p ≤ 0.05 (<i>Bmal1</i>), Student’s T-test.</p

<i>Per1</i> and <i>Per2</i> αODN attenuate the expression of corresponding transcripts in the SCN.

<p>2-h incubation of SCN slices with αODN results in a 45% decrease in <i>Per1</i> transcripts <b>(A)</b> and a 60% decrease in <i>Per2</i> transcripts <b>(B)</b> 4 h after initiation of treatment with the corresponding αODN. No change in GAPDH mRNA was evident following either treatment, which was used as a normalization control.</p

E-box decoy blocks binding at E-box sites in SCN 2.2 cells.

<p>Electromobility shift assay of an E-box probe incubated with nuclear extracts of SCN 2.2 cells transfected with 1 μM E-box decoy or missense ODN. Media lane indicates non-transfected control. Arrow = retarded mobility of the E-box probe. This DNA-protein interaction is absent in SCN 2.2 cells transfected with the E-box decoy up to 24 h (<i>n = 3</i>).</p

At CT 10, melatonin induces of <i>Per1</i> and <i>Per2</i> transcription by 120 min.

<p><b>A)</b> qPCR amplification products migrate at the predicted size and are distinguishable on an 8% polyacrylamide gel stained with ethidium bromide (<i>Per1 =</i> 113 bp, <i>Per2</i> = 90 bp, <i>BMAL1</i> = 79 bp). <b>B)</b> Melatonin has no significant effect on the expression levels of <i>Per1</i>, <i>Per2</i>, or <i>Bmal1</i> mRNA 30 min following the initiation of treatment (p ≥ 0.05, Student’s T Test). <b>C)</b> Melatonin treatment significantly increases <i>Per1</i> and <i>Per2</i>, but not <i>Bmal1</i>, transcripts, at 120 min. Data are shown as percent change of relative mRNA levels compared to control ± SEM, <i>n</i> = 3-4/condition. ***p ≤ 0.001 (<i>Per1</i>), *p ≤ 0.05 (<i>Per2</i>), p ≥ 0.05 (<i>Bmal1</i>), Student’s T-test.</p

<i>Per1</i> is required for melatonin to alter the phase of SCN neuronal activity rhythms at CT 10.

<p><b>A)</b> The spontaneous electrical activity rhythm in SCN brain slices peaks at CT 6.38 ± 0.13 in controls. The dotted line indicates the mean time-of-peak for untreated slices. Long, vertical boxes represent subjective night, CT 12–14. <b>B)</b> At CT 10, MEL (1 nM, 10 min) advances the electrical activity rhythm by 3.6 h ± 0.10 (<i>n = 3</i>). Arrow = time of melatonin treatment. <b>C)</b> <i>Per1</i> αODN application from CT 8–10 has no significant effect on the time-of-peak electrical activity (<i>n = 3</i>). Small box = duration of ODN exposure. <b>D)</b> The MEL-induced phase advance is completely blocked by <i>Per1</i> αODN (<i>n = 3</i>). <b>E)</b> <i>Per1</i> missense ODN has no effect on the MEL-induced advance in time-of-peak electrical activity (<i>n = 3</i>). <b>F)</b> <i>Per1</i> missense ODN does not block the MEL-induced phase advance at CT 10. <b>G)</b> Summary of the effects of <i>Per1</i> ODN on MEL-induced phase advances at CT 10. **indicates statistically significant difference compared to controls (p ≤ 0.001) as determined by 1-way ANOVA with Tukey’s <i>post-hoc</i> analysis.</p

Functional Peptidomics: Stimulus- and Time-of-Day-Specific Peptide Release in the Mammalian Circadian Clock

Daily oscillations of brain and body states are under complex temporal modulation by environmental light and the hypothalamic suprachiasmatic nucleus (SCN), the master circadian clock. To better understand mediators of differential temporal modulation, we characterize neuropeptide releasate profiles by nonselective capture of secreted neuropeptides in an optic nerve horizontal SCN brain slice model. Releasates are collected following electrophysiological stimulation of the optic nerve/retinohypothalamic tract under conditions that alter the phase of the SCN activity state. Secreted neuropeptides are identified by intact mass via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found time-of-day-specific suites of peptides released downstream of optic nerve stimulation. Peptide release was modified differentially with respect to time-of-day by stimulus parameters and by inhibitors of glutamatergic or PACAPergic neurotransmission. The results suggest that SCN physiology is modulated by differential peptide release of both known and unexpected peptides that communicate time-of-day-specific photic signals via previously unreported neuropeptide signatures

Active Antioxidizing Particles for On-Demand Pressure-Driven Molecular Release

Overproduced reactive oxygen species (ROS) are closely related to various health problems including inflammation, infection, and cancer. Abnormally high ROS levels can cause serious oxidative damage to biomolecules, cells, and tissues. A series of nano- or microsized particles has been developed to reduce the oxidative stress level by delivering antioxidant drugs. However, most systems are often plagued by slow molecular discharge, driven by diffusion. Herein, this study demonstrates the polymeric particles whose internal pressure can increase upon exposure to H₂O₂, one of the ROS, and in turn, discharge antioxidants actively. The on-demand pressurized particles are assembled by simultaneously encapsulating water-dispersible manganese oxide (MnO₂) nanosheets and green tea derived epigallocatechin gallate (EGCG) molecules into a poly­(lactic-<i>co</i>-glycolic acid) (PLGA) spherical shell. In the presence of H₂O₂, the MnO₂ nanosheets in the PLGA particle generate oxygen gas by decomposing H₂O₂ and increase the internal pressure. The pressurized PLGA particles release antioxidative EGCG actively and, in turn, protect vascular and brain tissues from oxidative damage more effectively than the particles without MnO₂ nanosheets. This H₂O₂ responsive, self-pressurizing particle system would be useful to deliver a wide array of molecular cargos in response to the oxidation level

Quantitative Peptidomics for Discovery of Circadian-Related Peptides from the Rat Suprachiasmatic Nucleus

In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, 10 endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (adjusted <i>p</i>-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥30% increases at ZT 18, and the VGRPE­WWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN